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Clinical and Diagnostic Laboratory Immunology, October 2005, p. 1231-1234, Vol. 12, No. 10
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.10.1231-1234.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of LIAISON Treponema Screen, a Novel Recombinant Antigen-Based Chemiluminescence Immunoassay for Laboratory Diagnosis of Syphilis

Antonella Marangoni,1 Vittorio Sambri,1* Silvia Accardo,1 Francesca Cavrini,2 Antonietta D'Antuono,3 Alessandra Moroni,1 Elisa Storni,2 and Roberto Cevenini1

Microbiology Section,1 Dermatology Section, DMCSS, University of Bologna,3 Centro di Riferimento Regionale per le Emergenze Microbiologiche, Ospedale Policlinico S. Orsola, Bologna, Italy2

Received 30 May 2005/ Returned for modification 22 June 2005/ Accepted 29 July 2005


   ABSTRACT
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The purpose of this study was to evaluate the diagnostic performance of LIAISON Treponema Screen (DiaSorin, Saluggia, Italy), a new automated chemiluminescence immunoassay (CLIA), in comparison with that of rapid plasma reagin (RPR) and the following currently used treponemal tests: hemagglutination test (TPHA), immunoenzymatic assay (EIA), and Western blot (WB). First, a retrospective study was performed with a panel of 2,494 blood donor sera, a panel of 131 clinical and serologically characterized syphilitic sera, and 96 samples obtained from subjects with potentially interfering diseases or conditions. A prospective study was also performed by testing 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna, Italy, for routine screening for syphilis. As expected, RPR was the least specific method, especially when potentially cross-reacting sera were tested. On the contrary, all of the treponemal tests proved to be very specific (99.9%) and they performed with the following sensitivities: 100% (WB), 99.2% (CLIA), 95.4% (EIA), and 94.7% (TPHA).


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Syphilis, which is caused by the spirochete Treponema pallidum subsp. pallidum, is a chronic bacterial infection that remains a public health concern worldwide. Although syphilis rates are quite low in industrialized countries, the World Health Organization estimated that 12 million new cases of venereal syphilis occurred in 1999, more than 90% of them in developing countries, with a rapidly increasing number of cases in Eastern Europe (12, 17). Nevertheless, recent outbreaks have been reported in several cities in Europe and North America among men who have sex with men (1). The serologic detection of specific antibodies to T. pallidum is of particular importance in the diagnosis of syphilis, since the natural course of the infection is characterized by periods of latency (9, 16). Serologic tests are divided into nontreponemal and treponemal tests, and neither alone is sufficient for a correct diagnosis, since nontreponemal tests can be used to monitor therapy, but due to the low specificity the positive results obtained by these methods need to be confirmed by treponemal tests. On the contrary, since the positivity by treponemal tests lasts a lifetime, treponemal tests cannot be useful for the follow-up of patients. Consequently, the quest for new, simple, reliable, and money-saving diagnostic methods continues. Nontreponemal tests include the Venereal Disease Research Laboratory test and the rapid plasma reagin (RPR) card test. Treponemal tests include the serum fluorescent treponemal antibody absorption test, the T. pallidum hemagglutination (TPHA) test, the immunoenzymatic assay (EIA), and the Western blot (WB) test; both the EIA and the WB test can be based on either whole-cell lysate (6, 7, 15) or recombinant (2, 13, 14, 19, 20) treponemal antigens. The purpose of this study was to evaluate diagnostic performances of LIAISON Treponema Screen (DiaSorin, Saluggia, Italy), a new automated chemiluminescence immunoassay (CLIA), in comparison with performances of EIA, WB, and TPHA. First, a retrospective study was performed with a panel of 2,494 blood donor sera, a panel of 131 clinical and serologically characterized syphilitic sera, and 96 samples obtained from persons with diseases or conditions that may cause false-positive reactions in tests for syphilis. A prospective study was also performed by testing 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna, Italy, for routine screening for syphilis.

For the retrospective study, sera were obtained from three different groups of subjects. The first group of 2,494 specimens included negative controls, and it was obtained from two blood donor banks (of the Etablissement Français du Sang [EFS]) in France (996 from EFS, Tours, and 1,498 from EFS, Lille). A second source of sera was a group of 131 patients suffering from different stages of syphilis who attended the sexually transmitted disease outpatient clinic of the St. Orsola Hospital in Bologna, Italy, and the Department of Preventive Pediatrics and Neonatology, University of Bologna, Italy. The staging of the disease was done by following the clinical and laboratory criteria proposed by Norris and Larsen (11). In particular, 38 samples were from patients suffering from untreated early syphilis (7 primary syphilis cases and 31 secondary syphilis patients), 77 specimens were from patients with previously treated latent disease, and 11 sera were obtained from subjects with untreated late syphilis (5 cardiovascular syphilis cases and 6 patients with neurological involvement). Finally, five specimens were drawn from newborns with a clinical diagnosis of highly probable congenital syphilis, according to CDC classification (3): all of these babies were born from untreated mothers, and at birth they had a quantitative nontreponemal serologic titer fourfold greater than the mother's titer.

Furthermore, a panel of 96 sera was obtained from subjects with some of the most common biological conditions possibly resulting in false-positive reactivity in syphilis serology. In this group, the following specimens were included: 30 serum samples obtained from patients with culture-confirmed Lyme disease, 10 serum samples from patients suffering from Streptococcus pyogenes acute infection (streptolysin O antibody response of >400 IU/ml), 10 serum samples drawn from subjects with a clinical diagnosis of infectious mononucleosis detected as positive by the Paul-Bunnel-Davidsohn agglutination, 5 specimens from pregnant women, and 26 from subjects suffering from autoimmune disorders (16 antinuclear antibody-positive sera and 10 rheumatoid factor-positive sera). Finally, 15 sera were obtained from patients with a severe periodontal situation; all of the crevicular fluid samples obtained from these patients were Treponema denticola positive when tested by real-time PCR carried out using a LightCycler system, and all of the sera were reactive when tested by a "homemade" T. denticola EIA (data not shown).

For the prospective study, 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna for routine screening for syphilis were evaluated.

LIAISON Treponema Screen (DiaSorin, Saluggia, Italy) is a qualitative, fully automated method for determination of specific antibodies to T. pallidum in human serum or plasma. This new method is a one-step sandwich CLIA. Recombinant specific T. pallidum antigen TpN17 is used for coating magnetic particles (solid phase), and the same antigen is linked to an isoluminol derivative (isoluminol-antigen conjugate). During the incubation, antibodies to T. pallidum present in calibrators, samples, or controls bind to the solid phase and to the antigen conjugate. The unbound material is removed with a wash cycle. Subsequently, the starter reagents are added and a flash chemiluminescence reaction is thus induced. The light signal, and hence the amount of isoluminol-antibody conjugate, is measured by a photomultiplier as relative light units and is indicative of antibodies to T. pallidum concentration present in calibrators, samples, or controls. The global results have been reported in the package insert upon conversion of arbitrary units (AU) per ml into index units (3 AU/ml corresponds to an index of 1). The results were evaluated using a cutoff value of an index of 1.0 with a gray zone of ±10% and interpreted as follows. Samples scored with an index of <0.9 were considered negative, and samples with an index of ≥1.1 were considered positive. Finally, samples showing a value falling in the gray range (i.e., 0.9 < index < 1.1) were boundary cases, and following the manufacturer's instructions, they were tested again.

Source, propagation, and extraction of T. pallidum subsp. pallidum, Nichols strain, were previously reported (10). Separation of T. pallidum polypeptides was performed with a Laemmli buffer system by using a 12% wt/vol acrylamide gel (8, 10). The WB procedure was performed according to Towbin et al. (18). After electrophoretic transfer, the blots were incubated overnight at room temperature with sera diluted 1:100 in phosphate-buffered saline containing 0.05% (vol/vol) Tween 20. Antigen-antibody complexes were detected by using a peroxidase-labeled rabbit antiserum to human immunoglobulin G (DAKO, Copenhagen, Denmark) diluted 1:500 in phosphate-buffered saline containing 0.05% (vol/vol) Tween 20 and 4-chloro-1-naphthol (Bio-Rad), as already described (10). A WB test was considered positive when at least three bands out of TpN47, TmpA, TpN17, and TpN15 were clearly recognized. A test was considered negative when no bands or bands for fewer than the three above-mentioned T. pallidum antigens were recognized (14).

The following commercial serologic tests were used: syphilis screening recombinant EIA (Radim, Pomezia, Italy), set up with recombinant forms of TpN17 and TpN15; TPHA (AlfaWasserman, Milan, Italy); and RPR (Radim, Pomezia, Italy). Testing was performed by following the manufacturer's instructions. Titers of ≥80 were considered positive for TPHA testing.

Preliminary experiments were done by testing the 2,494 negative control sera obtained from two French blood banks with LIAISON Treponema Screen. All of the sera were also tested by TPHA, syphilis screening recombinant EIA, and WB. No serum showed a titer of ≥80 when tested by TPHA. On the contrary, one sample was equivocal and two were scored as positive by LIAISON Treponema Screen. One of these two reactive sera was also positive by WB and EIA. No serum showing discrepant results was RPR reactive; on the contrary, (of 69 2,491) samples that scored as negative by all the treponemal tests were RPR positive. In order to assess the specificity of the new CLIA, we also tested 96 sera obtained from patients suffering from potentially cross-reactive conditions (Table 1). No serum gave a positive result when tested by CLIA, EIA, or WB. On the contrary, one sample obtained from a Lyme disease patient showed a TPHA titer of 80. This sample, drawn from a patient with no history of syphilis, was negative when tested by RPR. In this group of 96 serum samples, 6 samples were RPR reactive.


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  		TABLE 1. RPR and treponemal test reactivities detected in the panel of negative control seraa

 
Results obtained by all of the diagnostic methods when syphilis sera were tested are summarized in Table 2. Sensitivities and specificities of all methods are reported in Table 3. WB and CLIA were the most sensitive tests, especially when primary and latent syphilis sera were tested (Table 2).


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  		TABLE 2. Test reactivities in relation to stage of syphilis and treatment status

 

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  		TABLE 3. Sensitivities and specificities of tests, as detected in the retrospective study

 
For the prospective study, 1,800 unselected sera submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna for routine screening for syphilis were tested. Eighty-two samples out of this group of sera were positive and one was equivocal when tested by LIAISON Treponema Screen. All of these 83 sera were WB positive. One extra WB-positive serum was found in the group of 1,800 sera. A TpN17 band was faintly recognized, but the remaining three diagnostic bands were strongly reactive. The overall agreement between CLIA and WB was 99.9%, whereas the agreement between EIA and CLIA was 98.7%. Compared to TPHA, CLIA performed with an agreement of 99.3%. Seventy-four samples scored as positive by RPR: 35 were WB reactive, whereas the remaining 39 were negative when tested by all of the treponemal tests.

The diagnosis of syphilis is based upon clinical symptoms, laboratory examination of skin or mucous lesion material, and serology (9). Since lesion material is currently available for examination only during the early stages of disease, the main laboratory diagnostic tool for older stages of syphilis is the detection of specific antibody response to T. pallidum (11, 16). Recently, several different preparations of recombinant T. pallidum antigens have been described and their diagnostic performances and reliabilities have been reported (2, 19, 13, 14, 20). Chemiluminescent immunoassays have been evaluated in past years (4, 5), and this technique showed to be at least as sensitive and specific as the immunoenzymatic technique. Moreover, CLIA is very simple to perform and cost saving. In the present study, the diagnostic performances of a new chemiluminescent assay, LIAISON Treponema Screen, were compared with those obtained from the immunoenzymatic assay routinely used in our laboratory, syphilis screening recombinant EIA, and with the results obtained from two different confirmatory methods, WB and TPHA. Moreover, since many laboratories still use a nontreponemal test for screening, a comparison between LIAISON Treponema Screen and RPR results was also performed. So far, no other CLIA has been developed for the diagnosis of syphilis, so two different approaches were chosen to verify the sensitivity and specificity of this new test. At first, a retrospective study was performed with a panel of 2,494 blood donor sera, 131 syphilitic sera, and 96 samples obtained from persons with diseases or conditions that may cause false-positive reactions in syphilis serology. LIAISON Treponema Screen proved to be a very good alternative to the traditional immunoassays, since it was found to be as specific (99.9%) as the other treponemal tests; moreover, it showed a higher sensitivity (99.2%) than EIA (95.4%) and TPHA (94.7%), especially when primary syphilis samples were tested. Only WB was 100% sensitive. LIAISON Treponema Screen was not only more specific than RPR, as expected, but also more sensitive when samples from early syphilis patients were tested.

A prospective study was also performed by testing 1,800 unselected samples submitted to the Microbiology Laboratory of the St. Orsola Hospital in Bologna for routine screening for syphilis. Considering WB as the "gold standard" method, the agreement between LIAISON Treponema Screen and WB was 99.9%.

We conclude that the high sensitivity and specificity of LIAISON Treponema Screen and its suitability for automation make it an ideal screening test.


   FOOTNOTES
 
* Corresponding author. Mailing address: Section of Microbiology, DMCSS, University of Bologna, St. Orsola Hospital, via Massarenti 9, 40138 Bologna, Italy. Phone: 39 051 4290 913. Fax: 39 051 636 4516. E-mail: vsambri{at}med.unibo.it. Back


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Clinical and Diagnostic Laboratory Immunology, October 2005, p. 1231-1234, Vol. 12, No. 10
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.10.1231-1234.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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